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Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.
- Source :
-
Journal of molecular biology [J Mol Biol] 2009 Jan 16; Vol. 385 (2), pp. 393-404. Date of Electronic Publication: 2008 Nov 12. - Publication Year :
- 2009
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Abstract
- Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.
- Subjects :
- Adenosine Diphosphate metabolism
Adenosine Monophosphate metabolism
Adenosine Triphosphatases genetics
Adenosine Triphosphatases metabolism
Adenosine Triphosphate metabolism
Amino Acid Sequence
Amino Acid Substitution
DNA, Single-Stranded metabolism
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation, Missense
Protein Binding
Recombination, Genetic
Sequence Alignment
Bacteriophage T4 enzymology
Catalytic Domain genetics
DNA-Binding Proteins genetics
DNA-Binding Proteins metabolism
Histidine genetics
Membrane Proteins genetics
Membrane Proteins metabolism
Viral Proteins genetics
Viral Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1089-8638
- Volume :
- 385
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 19027026
- Full Text :
- https://doi.org/10.1016/j.jmb.2008.11.003